Measurement of <em>In Vitro</em> Integration Activity of HIV-1 Preintegration Complexes

Abstract

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested realtime quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration

“…To test this, we compared the integration activity in vitro of PICs extracted from cells inoculated with the WT or RK virus. Due to the well-recognized technical challenges associated with isolating the nuclear PICs, we used the cytosolic extracts from Jurkat or SupT1 cells 74, 131, 132 . The integration activity of these PICs is the measure of the number of viral DNA copies integrated into a heterologous target DNA and is quantified via a nested PCR assay.…”

HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification

“…Therefore, we employed a biochemical approach involving the extraction of PICs from acutely infected cells and quantified viral DNA integration activity in vitro (SI-Fig. 1A-B) 72–74 . We coupled PIC-VDI with biochemical assembled nucleosomes to define target substrate preference for HIV-1 DNA integration.…”

“…The in vitro integration assays were performed using a modified version of a protocol detailed by Balasubramaniam and colleagues 72–74 . The in vitro integration reaction was carried out by mixing 50 μl of PIC with 300 ng of the indicated target DNA, then allowing the mixture to incubate at 37°C for 45 minutes.…”

The Role of Capsid in HIV-1 Nuclear Entry

“…Traditionally, reverse transcription was thought to complete in the cytoplasm prior to nuclear entry as integration-competent PICs could be isolated from cytoplasmic fractions [ 19 , 20 , 21 , 22 ]. Additionally, premature uncoating triggered anti-viral responses due to exposure of viral cDNA to cytoplasmic sensors [ 24 , 25 , 26 , 27 ].…”

“…Biochemical analysis of pre-integration complexes (PICs) found little CA present [ 12 ] and particles were only sensitive to tripartite motif-containing protein 5α (TRIM5α), a host restriction factor that targets the CA lattice, for approximately an hour after cell entry [ 13 ]. Furthermore, uncoating was shown to be dependent on reverse transcription [ 14 , 15 , 16 , 17 , 18 ], which was believed to mainly take place in the cytoplasm, as integration-competent PICs could be isolated from cytoplasmic fractions [ 19 , 20 , 21 , 22 ]. Using GFP as a fluid phase marker in virions, and following infections by live-cell imaging, Mamede et al endorsed an early uncoating model.…”